Receptor activator of NF-?B ligand (RANKL) is a TNF?-type cytokine that is produced by mesenchymal lineage cells such as chondrocytes, osteoblasts and osteocytes, regulated by a number of local inflammatory mediators including TNF?, IL-1 and IL-6 and systemic regulators exemplified by 1,25(OH)2D3 and PTH, and known to play an essential role in bone formation and skeletal remodeling. This cytokine also modulates the immune system and is essential for a pleiotropic collection of additional biologic activities as well. RANKL also plays important underlying roles in bone loss diseases of various etiologies, in chronic inflammatory and autoimmune syndromes and in cancer development and progression. These diverse biological roles support the idea that TNFSF11 (the RANKL gene) is exquisitely regulated, and indeed, our recent work and that of others highlights this hypothesis. Accordingly, a complex set of at least ten upstream distal Tnfsf11 enhancers integrates both factor dependent and cell-specific regulation in osteoblasts as well as T and perhaps B cells. The importance of the Tnfsf11 gene as a basic regulatory paradigm coupled with its significant biological importance in the skeleton and elsewhere prompts the following specific aims. Aim 1: To define important epigenetic, structural and functional determinants within the Tnfsf11 locus which are essential for both transcription factor-dependent and cell type-specific expression of RANKL within the skeleton. Aim 2: To assess the consequence of individual Tnfsf11 enhancer deletions within the mouse genome on bone cell RANKL expression and regulation and on the skeletal phenotypes that emerge following physiological perturbations associated with age, altered mineral homeostasis, and physiologic state (lactation) in vitro and in vivo. RANKL plays a central role in bone formation during growth, in skeletal remodeling in the adult and in bone repair following fracture. Aberrant expression of RANKL as a result of disturbances in endocrine function, chronic inflammatory diseases such as arthritis or tumor progression are known to result in site-specific bone loss and/or osteoporosis or to produce other aberrant tissue/disease phenotypes. Our present studies are designed to determine components and mechanisms responsible for factor-specific and cell typeselective expression of RANKL in the skeleton and in immune cells in vitro and in vivo. These insights may identify useful therapeutic strategies for controlling RANKL expression, thus ameliorating the bone loss associated with osteoporosis, autoimmune syndromes or other related diseases.